Glioblastomas are aggressive primary brain cancers that recur as therapy-resistant tumors. Myeloid cells control glioblastoma malignancy, but their dynamics during disease progression remain poorly understood. Here, we employed single-cell RNA sequencing and CITE-Seq to map the glioblastoma immune landscape in newly diagnosed and recurrent patients and in mouse tumors. This revealed a large and diverse myeloid compartment, with dendritic cell and macrophage populations that were conserved across species and were dynamic across disease stages. Tumor-associated macrophages (TAMs) consisted of microglia-or monocyte-derived populations, with both exhibiting additional heterogeneity, including subsets with conserved lipid and hypoxic signatures. Microglia-and monocytederived TAMs (Mo-TAMs) were self-renewing populations that competed for space and could be depleted via CSF1R blockade. Microglia-derived TAMs were predominant in newly diagnosed tumors but were outnumbered by Mo-TAMs upon recurrence, especially in hypoxic tumor environments. Our results unravel the glioblastoma myeloid landscape and provide a framework for future therapeutic interventions.

Daratumumab is a CD38‐targeted human monoclonal antibody with direct anti‐myeloma cell mechanisms of action. Flow cytometry in relapsed and/or refractory multiple myeloma (RRMM) patients treated with daratumumab revealed cytotoxic T‐cell expansion and reduction of immune‐suppressive populations, suggesting immune modulation as an additional mechanism of action. Here, we performed an in‐depth analysis of the effects of daratumumab on immune‐cell subpopulations using high‐dimensional mass cytometry. Whole‐blood and bone‐marrow baseline and on‐treatment samples from RRMM patients who participated in daratumumab monotherapy studies (SIRIUS and GEN501) were evaluated with high‐throughput immunophenotyping. In daratumumab‐treated patients, the intensity of CD38 marker expression decreased on many immune cells in SIRIUS whole‐blood samples. Natural killer (NK) cells were depleted with daratumumab, with remaining NK cells showing increased CD69 and CD127, decreased CD45RA, and trends for increased CD25, CD27, and CD137 and decreased granzyme B. Immune‐suppressive population depletion paralleled previous findings, and a newly observed reduction in CD38 + basophils was seen in patients who received monotherapy. After 2 months of daratumumab, the T‐cell population in whole‐blood samples from responders shifted to a CD8 prevalence with higher granzyme B positivity ( P = 0.017), suggesting increased killing capacity and supporting monotherapy‐induced CD8 + T‐cell activation. High‐throughput cytometry immune profiling confirms and builds upon previous flow cytometry data, including comparable CD38 marker intensity on plasma cells, NK cells, monocytes, and B/T cells. Interestingly, a shift toward cytolytic granzyme B + T cells was also observed and supports adaptive responses in patients that may contribute to depth of response. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

CD38-targeted antibody, daratumumab, is approved for the treatment of multiple myeloma (MM). Phase 1/2 studies GEN501/SIRIUS revealed a novel immunomodulatory mechanism of action (MOA) of daratumumab that enhanced the immune response, reducing natural killer (NK) cells without affecting efficacy or safety. We further evaluated daratumumab’s effects on immune cells in whole blood samples of relapsed/refractory MM patients from both treatment arms of the phase 3 POLLUX study (lenalidomide/dexamethasone [Rd] or daratumumab plus Rd [D-Rd]) at baseline (D-Rd, 40; Rd, 45) and after 2 months on treatment (D-Rd, 31; Rd, 33) using cytometry by time-of-flight. We confirmed previous reports of NK cell reduction with D-Rd. Persisting NK cells were phenotypically distinct, with increased expression of HLA-DR, CD69, CD127, and CD27. The proportion of T cells increased preferentially in deep responders to D-Rd, with a higher proportion of CD8+ versus CD4+ T cells. The expansion of CD8+ T cells correlated with clonality, indicating generation of adaptive immune response with D-Rd. D-Rd resulted in a higher proportion of effector memory T cells versus Rd. D-Rd reduced immunosuppressive CD38+ regulatory T cells. This study confirms daratumumab’s immunomodulatory MOA in combination with immunomodulatory drugs and provides further insight into immune cell changes and activation status following daratumumab-based therapy.

The VirVarSeq is available, together with a user's guide and test data, at sourceforge: http://sourceforge.net/projects/virtools/?source=directory.